Co-existence of two plasmids harboring transferable resistance-nodulation-division pump gene cluster, tmexCD1-toprJ1, and colistin resistance gene mcr-8 in Klebsiella pneumoniae

Background The emergence of plasmid-mediated mobile colistin resistance (mcr) gene poses a great challenge to the clinical application of polymyxins. To date, mcr-1 to mcr-10 have been found in animals, humans, and the environment. Among them, mcr-8 was first identified in Klebsiella pneumoniae (K. pneumoniae) of swine origin, and then mcr-8.1 to mcr-8.5 were successively identified. Notably, K. pneumoniae is the major host of the mcr-8 gene in both animals and humans. This study aims to explore the characteristics of K. pneumoniae strains carrying the mcr-8 gene and tmexCD1-toprJ1 gene cluster and investigate the correlation between these two antibiotic resistance genes. Methods The isolates from the poultry farms and the surrounding villages were identified by mass spectrometer, and the strains positive for mcr-1 to mcr-10 were screened by polymerase chain reaction (PCR). The size of the plasmid and the antimicrobial resistance genes carried were confirmed by S1-nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern hybridization, and the transferability of the plasmid was verified by conjugation experiments. Antimicrobial susceptibility testing (AST) and whole genome sequencing (WGS) were used to characterize the strains. Results Two K. pneumoniae isolates (KP26 and KP29) displaying polymyxin resistance were identified as mcr-8 gene carriers. Besides that, tigecycline-resistant gene cluster tmexCD1-toprJ1 was also found on the other plasmid which conferred strain resistance to tigecycline. Through epidemiological analysis, we found that the mcr-8 gene has dispersed globally, circulating in the human, animals, and the environment. Furthermore, our analysis suggests that the coexistence of mcr-8 and tmexCD1-toprJ1 on a single plasmid might evolved through plasmid recombination. Conclusions Although the mcr-8 and tmexCD1-toprJ1 gene clusters in the two strains of K. pneumoniae in this study were on two different plasmids, they still pose a potential threat to public health, requiring close monitoring and further study. Supplementary Information The online version contains supplementary material available at 10.1186/s12941-024-00727-x.


Background
Multi-drug resistant (MDR) pathogens have become a vital global issue that caused by the overuse of antimicrobials in recent years [1].Among MDR pathogens, Klebsiella pneumoniae is considered as the notorious recipient of multiple antimicrobial resistance (AMR) genes as hundreds of mobile AMR genes have been found in this bacterium [2].Accumulation of AMR genes in K. pneumoniae is primarily the result of horizontal gene transfer (HGT) driven by plasmids and mobile genetic elements.The transmission of AMR genes in bacteria has posed a great threat to clinical and public health problems [3].
Polymyxins are a family of cyclic lipopeptides among which polymyxin B and polymyxin E (colistin) are the most used and studied variants.It acts as the last-resort antibiotics effective against infections caused by MDR Gram-negative bacteria in the clinical treatment.By electrostatic interacting with the negatively charged phosphate groups on lipid A of lipopolysaccharide (LPS) in the bacterial outer membrane, polymyxins inserted into the fatty acyl layer of the outer membrane and caused membrane disorganization and eventual cell death [4][5][6][7].Since Liu et al. [8] reported for the first time the emergence of the plasmid-mediated polymyxin resistance gene mcr-1 in Enterobacteriaceae, 10 mcr gene variants (mcr-1 to mcr-10) were found in isolates sourcing from animals, environment, and humans over 20 countries [9][10][11].The mcr gene mediates resistance by encoding a phosphoethanolamine (pEtN) transferase, which adds pEtN to the lipid A portion of LPS, thereby reducing the number of negatively charged phosphate groups on its surface.This modification results in a decreased affinity for polymyxin to bind to the target site, allowing for resistance to occur [10,12].A search of the GenBank database revealed that the mcr-8 gene was mainly present in K. pneumoniae of human and animal origin [13].Following the identification of a plasmid carrying mcr-8.2 in K. pneumoniae, it has been speculated that animalderived Klebsiella may be a reservoir for the mcr-8 gene [14].Gene mcr-8 was identified on the transferable IncII plasmid of K. pneumoniae, and the acquisition of a single mcr-8 gene significantly increased the colistin resistance of Escherichia coli (E.coli) and K. pneumoniae [13].
Tigecycline is a tetracycline class of antibacterial drugs that can be used to treat infections caused by polymicrobial MDR including both Gram-positive and Gram-negative bacteria [15].The emergence of tigecycline resistance has been reported worldwide though few studies are available for deciphering the molecular mechanism of resistance to tigecycline.Recently, a novel plasmid-harbored resistance-nodulation-division (RND) efflux pump gene cluster, tmexCD1-toprJ1, was reported to mediate tigecycline resistance in Gram-negative bacteria [16,17].The global dissemination of plasmid-borne drug resistance genes threatens to compromise the efficacy of polymyxins and tigecycline, increasing the difficulty of treatment of associated bacterial infections.
This study aimed to investigate the mechanism of plasmid-mediated colistin and tigecycline resistance by analyzing the prevalence and genetic characteristics of the mcr gene and tmexCD1-toprJ1 gene, underlining the cotransmission risk of these resistance genes.

Isolation and identification of strain and mcr genes
Samples of chicken manure, farm workers and healthy people were collected from chicken farms and their neighboring villages in Hangzhou.Fecal samples (≈1.0 g) were placed in 5 ml sterile Luria-Bertani broth and cultured overnight in an incubator at 37 ℃.The next day, the bacterial solution was coated on MacConkey agar for overnight culture.The MacConkey agar was repeatedly underlined to obtain a single colony.The species of bacteria were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and the mcr genes of all isolates were subsequently sequenced by PCR and Sanger sequencing using specific primers [18].Primer pairs used in this study were listed in Additional file 1: Table S1.

Antimicrobial susceptibility testing
In accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines, agar or broth microdilution is used to assess antimicrobial susceptibility.Microbroth dilution method was used to determine the MIC values of polymyxins (colistin and polymyxin B) and tigecycline, and agar dilution method was used to determine the MIC values of other antibiotics.Susceptibility results for all strains to tigecycline were interpreted according to the breakpoints of the European Committee for Antimicrobial Susceptibility Testing (EUCAST) (https:// eucast.org/ clini cal_ break points/), and susceptibility results for other antibiotics were interpreted according to the CLSI guidelines.The AST was carried out using E. coli ATCC 25922 as the quality control standard.

Conjugation experiments
To verify the transfer ability of plasmids carrying the mcr-8 gene or tmexCD1-toprJ1 gene cluster, we used strains carrying mcr-8 or tmexCD1-toprJ1 as donors, and sodium azide-resistant E. coli J53 and rifampicin-resistant EC600 as the recipient strains, respectively.Mueller-Hinton agar containing 200 mg/L sodium azide and 2 mg/L polymyxin B, 200 mg/L rifampicin and 4 mg/L tigecycline were used as the screening medium for transconjugants.We then selected transconjugants on Mueller-Hinton agar with sodium azide and polymyxin B, and detected the presence of the mcr-8 gene in the transconjugants by PCR and DNA sequencing.

S1-PFGE and southern hybridization
To identify the number and size of plasmids in K. pneumoniae carrying the mcr-8 gene, we performed the S1-PFGE and Southern hybridization experiments.Salmonella H9812 was used as control strain and size marker [19].After electrophoresis, it was hybridized with a digoxigenin-labeled mcr-8 probe to determine the location of the mcr-8 gene, and finally detected using the nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate (NBT-BCIP) color detection kit (Roche, catalog no.11745832910).

Characterization of mcr-8-harboring plasmids
Plasmid pKP26-mcr8 was of type IncFIA/IncFII with a total length of 101,185 bp and contained only one antimicrobial resistance gene, mcr-8.1.It was 100% identical to plasmid pKP29-mcr8.Therefore, we only describe the characterization of pKP26-mcr8.The mcr-8.1 gene on pKP26-mcr8 was associated with the genetic context of IS903B-orf-dgkA-baeS-copR-mcr-8.1-orf-IS903B.The pKP26-mcr8 was shown to be conjugative under laboratory conditions.We performed comparative genomic analysis based on DNA sequences of pKP26-mcr8 and five plasmids with different sources in NCBI (Fig. 2).The results showed that pKP26-mcr8, pKP46-mcr8 and pKP91 [13] isolated from animals had similar

Evolutionary analysis of isolates carrying mcr-8
We retrieved genome sequences of 138 mcr-8 positive K. pneumoniae from the NCBI database.Among these isolates, 122 strains had definite source.There are 63 strains isolated from animals, 56 strains from human, and 3 from environment (Fig. 3).Twenty strains did not have complete genome, so we constructed a phylogenetic tree consisting of 118 K. pneumoniae isolates, K. pneumoniae KP26 and KP 29 based on single nucleotide polymorphisms (SNPs) of core genomes.The 120 K. pneumoniae isolates were clustered in 3 clonal groups (Fig. 4).These isolates were identified in North America, Asia and Africa.Most of them were detected in China, followed by Thailand and Vietnam, indicating that Asia is the main prevalent continent for mcr-8 positive K. pneumoniae.The most common MLST type among the 114 strains was ST43 (17/114), followed by ST11 (16/114), which is also  the dominant epidemic strain in China [20].K. pneumoniae KP26 and KP29 were belong to cluster II and closed to KP_SAMN23139063.It is worth noting that isolates in cluster III were from different countries around world isolated from humans, animals and environment.This implies that mcr-8 positive K. pneumoniae are undergoing evolution during the process of dissemination.

Characterization of the tmexCD1-toprJ1-harboring plasmid
The plasmid type of pKP26-tmexCD1 was IncFIB(Mar)/ IncHI1B, with a length of 271,379 bp.Resistance genes include aph( 6)-Id, aph(3')-Ia, aph(3')-Ib, aadA1, aadA2b, armA, bla DHA-1 , msr(E), mph(E), cmlA1, qnrB4, sul1, and sul3.The sequence of pKP29-tmexCD1 was 100% identical to pKP26-tmexCD1.To investigate the core genetic environment of tmexCD1-toprJ1, we analyzed the tmexCD1-toprJ1-containing regions from four tmexCD1-toprJ1-bearing plasmids in Klebsiella spp.isolated from animals, humans and environment.The core genetic structures of tmexCD1-toprJ1 in Klebsiella spp.were divided into two types (Fig. 5).The type I genetic context is IS903B-strB-strA-tnpR-∆tnpA-toprJ1-tmexD1-tmexC1-tnfxB1-∆hp-IS26.Four tmexCD1-toprJ1-bearing plasmids (pHKU57_1, pKP26-tmexCD1, pKP46-3, and pKPT698-tmexCD) were belong to type I, and slightly different is that the pHKU57_1 lacks an IS903B upstream.They possessed two insertion sequence (IS) Fig. 3 Among all 138 isolates containing mcr-8 in the NCBI database, 131 of them had geographical distribution with well-defined locations.Different hosts of the isolates are marked with different colors, with red, yellow, blue and black indicating human, animal, environmental and unknown origin respectively.The number of isolates harboring mcr-8 in each country is plotted under the country name on a world map.And the world map was created using the corresponding map data of the R package ggplot2 v3.3.5 elements IS26 located on the flank of tmexCD1-toprJ1, which conferred the ability to acquire and mobilize tmexCD1-toprJ1, resulting the transmission in Klebsiella.
The core genetic environment of type II was strB-strA-tnpR-ΔtnpA-toprJ1-tmexD1-tmexC1-tnfxB1-hp in plasmid pHNAH8I-1 [16].It lacked the flanked IS26 element compared with type I, which indicates that the mobilization of tmexCD1-toprJ1 might be driven by IS26 from the plasmid pHNAH8I-1 [21].The IS elements play an important role in the spreading of tmexCD1-toprJ1.

Discussion
In this study, we found that two strains of K. pneumoniae isolated from chickens were identified as belonging to multi-drug resistant ST11 type and carried both the mcr-8 gene and the tmexCD1-toprJ1 gene cluster on two different plasmids.The complete genome sequences of two strains were obtained by WGS and compared with the genome of other K. pneumoniae isolates carrying the mcr-8 gene and tmexCD1-toprJ1 retrieved from NCBI database.Our results showed that mcr-8 gene is globally dispersed and has been circulating in the human, animals, and the environment, urging us to pay close attention to the convergence and co-transmission of mcr-8 and the tmexCD1-toprJ1 gene in K. pneumoniae.
In recent years, the discovery of mcr genes carried by plasmids has attracted a great deal of public attention.To date, a total of ten mcr genes (mcr-1 to mcr-10) have been identified [10].The mcr-8 gene was initially detected on an IncFII-type plasmid carrying carbapenemaseencoding gene bla NDM in swine-origin K. pneumoniae.Although mcr-8 gene and its variants have been identified in humans, animals, and environmental sources of K. pneumoniae, Raoultella ornithinolytica, and Klebsiella quasipneumoniae, K. pneumoniae is still considered as the primary host for the mcr-8 gene in both animals and human [13,14,22,23].In this study, we collected 112 strains of Enterobacteriaceae isolated from chicken manure and identified two mcr-8-positive K. pneumoniae strains.WGS revealed that these two strains also carried the tigecycline resistance gene cluster tmexCD1-toprJ1.The genetic context of mcr-8 in pKP26-mcr8 and pKP29-mcr8 is flanked with IS903B.This arrangement is consistent with genetic context of the mcr-8 gene in pKP91 [13].But in the environmental isolate pZZW20-88 K, a single IS903B is located upstream of the mcr-8 gene, while ISKpn26 is located downstream.Similarly, in the human isolate pMCR8_095845, ISKpn26 lies in the downstream region of mcr-8 gene.Therefore, we speculated that the insertion sequence flanked with mcr-8 gene is variable and both IS903B and ISKpn26 can mediate the transference of mcr-8 gene.
We also analyzed the nucleotide and protein sequences of genes that are reported to be involved in polymyxin resistance, including mgrB, phoP/phoQ, pmrA/pmrB, crrA/crrB, qseB/qseC, yciM and lpxM in KP26 and KP29.In addition to crrA/crrB genes which were not exist, two type of point mutations were found in the lpxM of KP26 and KP29 (I7N and S229T).Whether lpxM mutations affects the polymyxins resistance in KP26 and KP29 needs further investigation.
The tnfxB1-tmexCD1-toprJ1 gene cluster was found on the transposon Tn5393.There are two IS26 located upstream and downstream of this gene cluster, indicating that the tmexCD1-toprJ1 gene cluster was transferred via IS26 transposition [21].In addition, we found that pKP26-tmexCD1 contain a umuC gene serving as the integration site for the mobilization of tmexCD1-toprJ1 gene cluster mediated by int gene.However, attempts to transfer the plasmid carrying this gene cluster into EC600 through conjugation experiments or electroporation, were unsuccessful.We inferred that the tmexCD1-toprJ1-carrying plasmid might have a preference for the host.This might explain the widespread presence of this gene cluster in K. pneumoniae, which is rare in other Enterobacteriaceae species [24].Apart from chicken in this study, the tmexCD1-toprJ1 was also detected in ST6265 K. pneumoniae strains from patient sources [25].This result suggests that strains carrying tmexCD1-toprJ1 are capable of transmitting between human and animals.We also investigated the existence of iron-absorption virulence gene, invasive virulence gene and adhesive virulence gene in KP26 and KP29 through WGS analysis.However, hypervirulence genes were not found in these two strains.This indicates that KP26 and KP29 are not hypervirulent strains.
Although our study found that the mcr-8 and tmexCD1-toprJ1 gene were located on the two different plasmids, it has been reported that the mcr-8 gene and the tmexCD1-toprJ1 gene cluster can coexist on the same plasmid and undergo co-transfer during transmission.Plasmid recombination have occurred during the conjugation between the mcr-8-carrying plasmid and tmexCD1-toprJ1-carrying plasmid mediated by IS26 [26].Using colistin could accelerate the process of plasmid fusion and formation of tigecycline-resistant strains.Through phylogenetic analysis of KP26, KP29, and mcr-8-carrying K. pneumoniae strains from the NCBI database, it was observed that the co-occurrence of the tmexCD1-toprJ1 gene cluster was found in 35% (42/120) of the strains.Therefore, it is highly possible that IS26 can mediate the plasmid fusion between pKP26-tmexCD1 and pKP26-mcr8 to carry more antibiotic resistance genes.The emergence of isolates resistant to both colistin and tigecycline is inevitable due to the use of these drugs as last-resort antibiotic treatments.This, in turn, facilitates the spreading of mcr-8 and tmexCD1-toprJ1 within the same strain.

Fig. 1
Fig. 1 S1-PFGE profile (left) and Southern hybridization (right) analysis using mcr-8-specific probes to detect mcr-8-harboring isolates.Salmonella strain H9812 was used as a control strain and size marker.The names of the isolates are shown in the first line.The arrows indicated the locations of mcr-8 harboring plasmids according to the Southern hybridization experiment

pKP29- 4 637 Fig. 2
Fig. 2 Genetic features of the mcr-8-carrying plasmid in K. pneumoniae strain KP26 and KP29.The linear genetic environment surrounding mcr-8 is depicted.Arrows indicate the direction of transcription for each gene, and different colors represent different genes.The mcr-8 gene is shown in red, genes associated with mobile elements are shown in green, the three genes in the figure are shown in black, and hp and other functional genes are shown in yellow.Regions with high homology are represented by gray shading

Fig. 4 Fig. 5
Fig. 4 Phylogenetic tree generated from the core genome sequences of the mcr-8-harboring isolates identified in this study and other mcr-8-harboring K. pneumoniae isolates in the NCBI database.Each isolate has a sample identifier (ID), location, MLST, and host.The figure shows important antimicrobial resistance genes (ARGs) and major virulence-associated genes (VGs)

Table 2
Features of the isolatesa No resistance genes or plasmid replicon was found